The subject of the invention is compounds implicated in resistance to eutypa dieback in plants as well as agents for combatting this disease, for example in the grapevine.
At present eutypa dieback is the most serious disease of the grapevine. In fact, it is present in vineyards throughout the world; it leads to the premature death of many grapevines of the sensitive varieties and there exist no curative agents. The parasite responsible for eutypa dieback of the grapevine is an ascomycete fungus existing in a perfect form Eutypa lata (Carter M. V., 1955, Apricot gummosisxe2x80x94a new development J. Dep. Agric. South Aust. 59: 178-184) Tul and C. Tul. Syn; E armeniacae Hansf. and Carter) or an imperfect form Libertella blepharis (Samuel G, 1933 xe2x80x9cGummosisxe2x80x9d or xe2x80x9cDiebackxe2x80x9d in apricot trees J. Dep. Agric. South Aust. 36: 979-980) A. L. Smith (syn. Cytosporina sp.).
Eutypa dieback is certainly an ancient disease, even though the pathogenic agent was observed on the grapevine only in 1973 in Australia. In France the disease was identified with certainty by Bolay in 1977 in the region of Languedoc-Roussillon. From 1978, the observations have multiplied in the vineyards of France which show the expansion of eutypa dieback. The reasons for the spread of this dieback disease, which is endemic, have still not been clearly identified to-day; according to certain authors, this progression might be attributable to the modification of certain cultural practices.
The grapevine is not the only perennial ligneous plant which is host to this parasite. In fact, the signs of eutypa dieback have been observed in particular on trees and bushes such as the apricot, black currant, cherry, tamarind, almond, apple . . .
The disease affects the trunk and the xe2x80x9carmsxe2x80x9d of the grapevine, sites of development of the mycelium, and the herbaceous parts which, however, never harbour the parasite. The ascospores of the fungus, released by the asci of the perithecia situated on the rind of the contaminated grapevines, penetrate via the wounds resulting from pruning into the lumen of the vessels of the xylem, then germinate at several millimeters from the surface of the wound and colonize the xylem, the cambium and the phloem. The installation of the mycelium in the xe2x80x9cwoodxe2x80x9d of the trunk and the arms of the grapevine leads to the formation of an always well-defined, hard and sectorial, brown necrosis, which has an appearance of dry rot, a sign of a preferential degradation of the cellulose of the wood.
The plants affected by eutypa dieback show typical symptoms in the spring. In the most characteristic cases, the branches are emaciated with short internodes; the leaves of small size, often coiled or even deformed, have a slightly chlorotic appearance and sometimes small marginal necroses. The inflorescences seem to develop normally until flowering, but they may then necrose or give bunches of small grapes (fruit drop). The symptoms described often affect one arm but the disease can then spread to all of the grapevine, leading to the death of the latter.
The duration of the slow incubation of the fungus in the trunk and the arms (3 to 10 years) confers on this disease in insidious character. The expression of the symptoms on the herbaceous organs is uncertain depending on the year: a contaminated grapevine does not show symptoms regularly, certainly for reasons related to environmental factors and in particular to climatic conditions. Moreover, the existence of isolates of different aggressivity has been demonstrated, making still more difficult the recognition of the disease.
The destruction of the parasite in the trunk of the grapevines or the reduction of its expression is not possible to-day. In the absence of curative pest control measures, different prophylactic and preventive measures have been recommended to contain the disease. It is recommended, for example, to limit the pressure of the inoculum by removing old grapevines and the arms cut after the pruning operation. Awareness of the conditions of release and germination of the ascospores has led to several recommendations: prune late while taking account of the climatic conditions (dry and calm weather), avoid prunings causing large surface wounds, protect each pruning wound by the manual application of a fungicide to limit the germination of the ascospores. Finally, the affected stains may be restored by the operation of cutting them back severely, which consists of preserving a stem existing below the diseased part of the trunk and of removing the upper part.
It can be seen that eutypa dieback is certainly harmful because the present pest control agents, uniquely prophylactic and preventive, are restrictive, expensive and of limited scope.
The economic impact of this disease is considerable. The surveys conducted in France since 1988 show that the levels of symptoms are variable and depend on the variety of grapevine and, for a given grapevine, depend on the year and the regions, sometimes up to 50% of the grapevines being subjected to the disease. The most sensitive varieties include the Sauvignon blanc, the Cabernet Sauvignon, the Ugni blanc, the Cinsault and the Chein whereas the Merlot and the Sxc3xa9millon are the most tolerant.
A network of observations based on a rigorous statistical approach set up in the context of a European contract (xe2x80x9cEutypa dieback controlxe2x80x9d programme No. 8001-CT-91.205) focussed on 85000 grapevines cultivated in 11 wine-growing regions and on 10 varieties of grapevine. It made it possible to show that all the varieties exhibit symptoms of eutypa dieback but to very variable extents: 30 to 80% in certain regions of Greece, 5 to 10% in the Rioja Alta region (Spain, Tempranillo variety) and in Italy (Trebbiano variety) and less than 2%, even 1%, in the wine-growing areas of Portugal.
This disease may also entail a reduction of the expression of the aromatic quality in certain grapevine varieties. In addition, eutypa dieback requires the premature replacement of the dead varieties which causes the rejuvenation of the vineyard, which is detrimental to quality. It should also be noted that the parasitic fungus plays a pioneer role in the installation of parasitic apoplexy (Carignon C., 1991), fungal complex also responsible for a dieback of the grapevine.
This disease also has indirect effects on quality by modifying the planting of the vines in the vineyard because the sensitive varieties are little by little replaced by more tolerant varieties, while however respectively the restrictions of the registered designation of origin. Furthermore, if the harvest volume is maintained in spite of varieties missing to a high extent, the quality of the wines may be affected.
Consequently, it is clear that for the winegrowers wishing to maintain a regular production of quality wines, eutypa dieback is presently the most worrying disease.
The inventors have shown that eutypa dieback is associated with the synthesis of a compound of the family of the lipophilic acids, 4-hydroxy-3-(3-methylbut-3-en-1-ynyl) benzaldehyde, called eutypine (Tey-Rulh P. et al. Phytochemistry, vol. 30, No. 2, pp. 471-473, 1991) in some tissues of the host harbouring the parasitic fungus. This toxic molecule is synthesized by the parasitic fungus.
In order to control this disease the inventors have been concerned with the structure and physiology of eutypine when it is synthesized in the host by the parasite and have observed in plants and in particular in the plants resistant to eutypa dieback, the synthesis of degradation products of this toxic constituent.
By the expression xe2x80x9cplants resistant to eutypa diebackxe2x80x9d is meant in the context of the present application, the capacity of the plants contaminated by the parasite responsible to resist the harmful effects induced directly or indirectly by the presence of the parasite.
This type of resistance may also be explained as the capacity of the plants contaminated by the fungus responsible for eutypa dieback to tolerate the presence of this parasite without their development, including their growth and their physiology, being affected to an extent which would entail the dieback of the plants in question.
The inventors have demonstrated a metabolic pathway for eutypine within the plants contaminated by the parasite, resulting in the synthesis of products non-toxic for these plants; as a result, they have suggested agents for conferring on plants sensitive to eutypa dieback properties of resistance or, where appropriate, agents for increasing the resistance capacities already existing in certain plants towards eutypa dieback.
In this connection, the inventors have identified and characterized in particular a polypeptide with enzymatic activity of the reductase type capable of promoting the degradation of the toxic eutypine into non-toxic constituents including a compound called eutypinol.
The inventors have isolated and identified the eutypine reductase enzyme and have succeeded in characterizing the nucleotide sequence coding for this enzyme.
The nucleotide sequence coding for this enzyme thus constitutes a novel means for considering the production of plants resistant to eutypa dieback in conformity with the definition which was given previously of this resistance. For example, the characterization of this nucleotide sequence makes it possible to prepare transgenic plants capable of overproducing eutypine reductase and of increasing the metabolism of the eutypine produced in the host by the parasite.
Hence the subject of the invention is a nucleotide sequence, characterized in that it codes for a polypeptide with eutypine reductase activity, this polypeptide comprising the amino acid sequence represented in FIG. 1 (SEQ ID NO. 3 and NO. 4).
According to a particular embodiment of the invention, this nucleotide sequence is characterized in that it comprises the nucleotide sequence included between the nucleotides 22 and 975 of the nucleotide sequence shown in FIG. 1 (SEQ ID NO. 3 and NO. 4).
As a variant, such a sequence is characterized in that it corresponds to the sequence shown in FIG. 1 (SEQ ID NO. 3 and NO. 4), which is a complementary DNA sequence (cDNA) of the VRER gene coding for the eutypine reductase molecule.
The subject of the present invention is also a nucleotide sequence hybridizing with one of the sequences previously described under highly stringent conditions.
Such highly stringent conditions are the following: placing in contact with a solution containing SSC (2xc3x97), 1% SDS for 5 min at 25xc2x0 C., then twice for 15 min. at 45xc2x0 C., then in a solution containing SSC (0.2xc3x97), 1% SDS twice for 15 min. at 45xc2x0 C.
Alternatively, the nucleotide sequence of the invention may be defined in that it possesses a sequence homology of more than 75% with the nucleotide sequence included between the nucleotides 22 and 975 of the sequence shown in FIG. 1 (SEQ ID NO. 3 and NO. 4), or with the nucleotide sequence shown in FIG. 1 (SEQ ID NO. 3 and NO. 4).
Preferably, this homology is greater than or equal to 80% and advantageously greater than or equal to 90%.
The nucleotide sequences may advantageously be used in the procedures of genetic engineering to produce the compounds according to the invention.
In this connection, a sequence should advantageously be used which complies with one of the preceding definitions under the control of a homologous or heterologous promoter, and preferably under the control of a promoter suitable for expression in prokaryotic and/or eukaryotic cells.
Preferably, and depending on the level of eutypine reductase activity desired, recourse should be had to a strong promoter for expressing a nucleotide sequence according to the invention. A suitable promoter is for example the strong promoter 35S of the CaMV (EP-B-0131623) permitting expression in plant cells.
Where appropriate, the promoter used is combined with other regulatory sequences such as expression enhancers.
As examples of vectors, mention should be made of the binary vector pGA described by An, G et al., 1988, Binary Vectors, pp 1-19 in Plant Molecular Biology Manual A3 Kluwer Acad. Pub., Dordrecht, The Netherlands.
The application also relates to recombinant prokaryotic or eukaryotic cells, containing a nucleotide sequence complying with one of the definitions given above.
Valuable recombinant cells in the framework of the embodiment of the invention are for example bacterial cells, in particular E. coli or Agrobacterium tumefaciens cells. They may also be eukaryotic cells and, in particular, plant cells. As examples, recourse should be had to cells of plant seeds or to grapevine cells, in particular when it is desired to implement resistance to eutypa dieback in the grapevine.
Also included in the framework of the invention are a biological tissue or plant producing a polypeptide compound complying with one of the preceding definitions after transformation by a nucleotide sequence according to the invention. Preferably, the recombinant plant is the grapevine.
The invention also relates to seeds obtained from said plants as well as to a process for the production of these plants or these seeds.
Preferably, the cells, tissues and plants transformed in the context of the invention contain the nucleotide sequence integrated stably into their genome. Any suitable transformation procedure may be used, such as electroporation, bombardment with microprojectiles carrying DNA, for example, by means of a particle gun, the co-culture of explants in the presence of Agrobacterium tumefaciens. 
Such a production process for a plant or a seed expressing a polypeptide compound with eutypine reductase activity is characterized in that it comprises the steps of
a) transformation of a plant cell with a nucleotide sequence defined above or a vector defined above under conditions permitting the stable and functional expression of the protein with eutypine reductase activity encoded in the above-mentioned nucleotide sequence;
b) regeneration of plants from the transformed plant cell of step a) to obtain plants expressing the protein with eutypine reductase activity;
c) where appropriate, production of seeds from the modified plants obtained in step b).
The process of the invention may advantageously be implemented to produce plants of the grapevine family, but also to produce plants resistant to eutypa dieback when these plants are naturally sensitive to this disease, for example perennial ligneous plants such as the apricot, black currant, cherry, tamarind, almond or apple.
Moreover, the subject of the invention is the use of the nucleotide sequence such as defined in the preceding pages as selection agent, a detector of the transformation of cells by means of a defined nucleotide sequence whose expression is coupled to that of the sequence used as selection agent. In the context of this application the sequence or part of the sequence of the VRER gene or its cDNA may be used as selection gene for making it possible to identify cells, tissues or plants genetically transformed by a sequence of interest. In this case, the selection gene is transferred with the gene of interest to the cells that it is desired to transform. The sequence of the gene of interest and that of the selection gene are combined so that the integration and/or the expression of the one is linked to the integration and/or the expression of the other. The value of using the sequence of the invention as selection gene or sequence lies in its making it possible in particular to avoid certain problems linked to the dissemination of the genes, a problem encountered with the genes for resistance to antibiotics or herbicides. In the framework of this application, the transformed cells, tissues or plants may be selected by being placed in contact with eutypine or with any substrate capable of being reduced by the eutypine reductase produced by the selection gene.
The selection of the transformed cells, tissues or organisms which have integrated the selection gene with the aid of eutypine or any suitable substrate should be feasible by prior determination of the threshold sensitivity to eutypine of the cell of the tissue or plant considered. This determination should be made by procedures similar to those known for the determination of the sensitivity of a given species or organism to an antibiotic or herbicide.
Hence the subject of the invention is also a recombinant nucleic acid containing a nucleotide sequence according to the invention, used as selection agent in combination with a sequence of interest, said combination making possible the linked integration of the selection marker and the sequence of interest in a defined cell host.
Consequently, the framework of the invention includes a procedure for the detection of cells transformed by a defined sequence of interest comprising:
a) the transformation of the cells with a recombinant nucleic acid comprising the gene of interest and a selection gene comprising a nucleotide sequence coding for a polypeptide with eutypine reductase activity according to the invention,
b) the placing of the cells obtained in step a) in contact with a substrate for eutypine reductase,
c) the detection of a reduction reaction undergone by the substrate
d) where appropriate, the selection of the transformed cells giving rise to this reduction reaction.
Hence the subject of the invention is also a polypeptide compound with eutypine reductase activity, characterized in that it comprises the amino acid sequence described in FIG. 1 (SEQ ID NO. 3 and NO. 4).
According to this definition, the eutypine reductase activity observed is the capacity conferred on the host of the parasite responsible for eutypa dieback or increased by this polypeptide compound to degrade or metabolize eutypine to degradation products non-toxic for the reference host usually sensitive to the toxicity linked to the presence of eutypine.
Etypine or 4-hydroxy-3-(3-methylbut-3-en-1-ynyl) benzaldehyde is a lipophilis weak acid which has been demonstrated to be capable of being degraded to a hydroxylated derivative, 4-hydroxy-3-(3-methylbut-3-en-1-ynyl) benzylic alcohol, called eutypinol in the context of the present application, as a result of an enzymatic reaction for which the constituent NADPH is a specific co-factor when the reaction is performed in vitro.
In an environment permitting it, the polypeptide compound according to the invention is hence capable of carrying out this enzymatic reaction to metabolize eutypine to a non-toxic derivative, in particular to the form of eutypinol.
Where appropriate, the eutypine reductase activity may be observed on other aldehydic substrates.
The polypeptide compound previously described is characterized by the presence in its structure of an amino acid sequence, the sequence of which has been given in FIG. 1 (SEQ ID NO. 3 and NO. 4); if necessary, it may include additional groups the presence and/or nature of which depends for example on the cell host in which it is expressed.
A valuable compound according to the invention is also characterized in that it has a molecular weight of about 36 kDa, calculated from the coding sequence and measured on the purified protein.
According to a particular embodiment of the invention, the polypeptide compound is characterized in that it is encoded in a nucleotide sequence comprising the coding nucleotide sequence included between the nucleotides 22 and 175 (these end nucleotides being included) of the sequence shown in FIG. 1 (SEQ ID NO. 3 and NO. 4).
The subject of the invention also includes compounds modified with respect to the polypeptide compound previously defined. In particular, they may be polypeptide compounds comprising an amino acid sequence derived from the amino acid sequence described in FIG. 1 (SEQ ID NO. 3 and NO. 4), for example by addition, deletion or substitution of at least one amino acid residue contained in this sequence, the compound formed having a eutypine reductase activity according to the definition which is given above.
When the compound of the invention is obtained by deletion of at least one amino acid residue of the sequence of the polypeptide compound previously described, the deletion may extend to the elimination of one or more fragments unnecessary or inessential for the eutypine reductase activity of the compound complying with the definition of FIG. 1 (SEQ ID NO. 3 and NO. 4).
Similarly, when a derivative of the compounds described above is obtained by substitution of certain amino acid residues, they may be replaced by neutral residues or by residues having the same properties or by residues capable of promoting the desired reducing properties.
According to a variant of the embodiment of the invention, the polypeptide compound is characterized in that it is recognized by antibodies obtained against a polypeptide complying with the amino acid sequence shown in FIG. 1 (SEQ ID NO. 3 and NO. 4).
Such antibodies are for example polyclonal antibodies obtained by immunization of an animal, in particular a rabbit, with the polypeptide compound with eutypine reductase activity according to the invention and recovery of the antibodies formed from the rabbit serum.
Also included in the context of the invention is a polypeptide compound characterized in that its amino acid sequence exhibits a sequence homology of at least 50% with the amino acid sequence shown in FIG. 1 (SEQ ID NO. 3 and NO. 4).
By homology is meant the identity of the amino acids compared when the two amino acid sequences are compared or, in the absence of identity, the conservation of the properties of the original amino acid in the substituted amino acid.
Advantageously, the homology previously in question is equal to at least 70%, and preferably equal to at least 80, even 90%, in numbers of amino acid residues, this homology being evaluated by reference to the amino acid sequence shown in FIG. 1 (SEQ ID NO. 3 and NO. 4).
The polypeptide compounds according to the invention may be obtained by any suitable method and in particular may be isolated from plants, for example from the bean Vigna radiata (Mung Bean) or may also be synthesized chemically or prepared by genetic engineering in particular in recombinant cells.
Other plants may also be used to extract the compounds of the invention, such as the grapevine, the potato, the carrot, etc . . .